Compositions and methods for regulating autolytic processes in bacteria

ABSTRACT

A nucleic acid sequence required for regulating the autolytic activity of bacteria is provided. Also provided are polypeptides encoded by the gene or mutant gene as well as vector and host cells for expressing these polypeptides. Methods for identifying and using agents which interact with the gene or mutant gene or polypeptides encoded thereby to inhibit bacterial growth and infectivity are also provided.

INTRODUCTION

[0001] This application claims the benefit of priority from U.S.provisional application Serial Nos. 60/273,791, filed on Mar. 6, 2001;60/312,546, filed on Aug. 15, 2001; and 60/329,140, filed on Oct. 12,2001, whose contents are incorporated herein by reference in theirentireties.

[0002] This invention was made in the course of research sponsored bythe National Institute of Health (NIH Grant No. RO1-AI37142). The U.S.government may have certain rights in this invention.

BACKGROUND

[0003] Staphylococci are hardy and ubiquitous colonizers of human skinand mucous membranes and were among the first human pathogensidentified. These bacteria constitute a medically important genera ofmicrobes as they are known to produce two types of disease, invasive andtoxigenic.

[0004] Invasive infections are characterized generally by abscessformation affecting both skin surfaces and deep tissues. In addition,Staphylococcus aureus (S. aureus) is the second leading cause ofbacteremia in cancer patients. Osteomyelitis, septic arthritis, septicthrombophlebitis and acute bacterial endocarditis are also relativelycommon.

[0005] There are also at least three clinical conditions resulting fromthe toxigenic properties of Staphylococci. The manifestation of thesediseases result from the actions of exotoxins as opposed to tissueinvasion and bacteremia. These conditions include: Staphylococcal foodpoisoning, scalded skin syndrome and toxic shock syndrome.

[0006]S. aureus are non-mobile, non-sporulating gram-positive cocci0.5-1.5 im in diameter, that occur singly and in pairs, short chains,and irregular three-dimensional clusters. S. aureus can grow over a widerange of environmental conditions, but they grow best at temperaturesbetween 30° C. and 37° C. and at a neutral pH. They are resistant todesiccation and to chemical disinfection, and they tolerate NaClconcentrations up to 12%. It has been found that the growth of the S.aureus becomes unusually sensitive to a high-NaCl concentration bydecreasing the Ca²⁺ concentration in growth media allowing for autolysis(Ishikawa, Microbiology and Immunology, 2000: 44(2):97-104).

[0007] Humans constitute the major reservoir of the S. aureus bacteria.The cross sectional carriage rate in adults is 15 to 40 percent. Themucous membranes of the anterior nasopharynx are the principal site ofcarriage. Other sites include the axillae, the vagina, the perineum andoccasionally the gastrointestinal tract. Colonization by S. aureus maybe intermittent or persistent and is probably influenced by bothmicrobial and host factors as well as by the nature of the competingnon-Staphylococcal flora.

[0008] The frequency of S. aureus infections has risen dramatically inthe past 20 years. This has been attributed to the emergence of multipleantibiotic resistant strains and an increasing population of people withweakened immune systems. It is no longer uncommon to isolate S. aureusstrains which are resistant to some or all of the standard antibiotics.This has created a demand for both new anti-microbial agents anddiagnostic tests for this organism. Accordingly, there is a need forbetter understanding of factors which regulate infectivity and growth ofS. aureus.

[0009] Genes identified as involved in the infectivity and/or growth ofS. aureus include the ArlS regulator, involved in adhesion (Fournier andHooper, Journal of Bacteriology, 2000: 182(14):3955-64), the pbpC gene,which affects the rate of autolysis, (Pinho et al., Journal ofBacteriology, 2000: 182(4):1074-9), lytRS (Brunskill and Bayles, Journalof Bacteriology, 1996: 178(19):5810-2), lrgA and lrgB (Fujimoto et al.,Journal of Bacteriology, 2000: 182(17):4822-8), and lytM identified inautolysis-deficient mutants of S. aureus (Ramadurai and Jayaswal,Journal of Bacteriology, 1997: 179(11):3625-31).

[0010] A new genetic locus of S. aureus which regulates autolyticprocesses has now been identified. The gene at this locus, referred toherein as RAT, regulator of autolytic activity, which regulatesautolytic processes, has been cloned and sequenced. The phrase, Awhichregulates expression of polypeptides involved in autolytic processes@used herein means that the gene or polypeptide encoded by the genecontrols, modulates or regulates the expression of polypeptides involvedin autolytic processes such as autolytic enzymes (e.g., mureinhydrolase, cell wall hydrolase, glycylglycine endopeptidase),polypeptides involved in environmental signaling, the secretion ofautolysins or other autolytic processes. Further, it has been shown thata mutation at this site renders S. aureus more susceptible to lysis withantibiotics.

SUMMARY OF THE INVENTION

[0011] The present invention provides a new genetic locus of S. aureusand other bacteria that is involved in the control, modulation orregulation (these latter three terms are used as equivalents herein) ofautolytic activity and processes in bacteria. Examples of the gene, amutant of the gene, as well as the polypeptides encoded by the gene andthe mutant of the gene are also provided.

[0012] An object of the present invention is to provide nucleic acidsequences isolated from S. aureus and other bacteria which regulateautolytic activity in bacteria. The nucleic acid sequences referred toherein are the RAT gene (SEQ ID NO:1), the RAT mutant gene (SEQ ID NO:3)or fragments thereof. In a preferred embodiment, the nucleic acidsequence is the RAT gene (SEQ ID NO:1).

[0013] Another object of the present invention is to providepolypeptides encoded by the RAT gene and vectors and host cellscomprising nucleic acid sequences encoding these polypeptides. In apreferred embodiment, the polypeptide has the sequence of SEQ ID NO:2.The sequence of the polypeptide encoded by the mutant of the RAT gene isalso provided (SEQ ID NO:4).

[0014] Another object of the present invention is to provide a vectorwhich comprises a transposon element and a polynucleotide sequence whichencodes the RAT polypeptide (SEQ ID NO:2) or the RAT mutant polypeptide(SEQ ID NO:4) and host cells comprising this vector.

[0015] Another object of the present invention is to provide a method ofidentifying agents that modulate autolytic activity of S. aureus andother bacteria through interaction with the RAT gene (SEQ ID NO:1) orRAT mutant gene (SEQ ID NO:3) or polypeptides encoded by RAT or the RATmutant gene. These agents are expected to be useful in the inhibition ofgrowth of S. aureus and other bacteria and in the treatment of hostsinfected by S. aureus and other bacteria. These agents can be used aloneor in combination with an antibiotic such as penicillin to promote lysisof the bacteria.

[0016] Accordingly, another object of the present invention is toprovide methods for modulating autolytic activity of S. aureus and otherbacteria to inhibit their growth and infectivity by contacting thebacteria with an agent which interacts with the RAT gene (SEQ ID NO:1)or RAT mutant gene (SEQ ID NO:3) or polypeptides encoded thereby.

[0017] Yet another object of the present invention is to provideanti-bacterial agents which comprise a compound which prevents orinhibits infections by S. aureus or other bacteria via interaction withthe RAT gene (SEQ ID NO:1) or RAT mutant gene (SEQ ID NO:3) orpolypeptides encoded by RAT or the RAT mutant gene.

[0018] Another object of the present invention is to provide a kit foridentifying the presence of the RAT gene (SEQ ID NO:1) or RAT mutantgene (SEQ ID NO:3) or the polypeptides they encode (SEQ ID NO:2 or SEQID NO:4, respectively).

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 graphically demonstrates the effect that subinhibitoryconcentrations of penicillin have on the RAT mutant as compared towild-type.

[0020]FIG. 2 graphically represents the viability of the RAT mutant ascompared to wild-type by propidium iodide staining.

DETAILED DESCRIPTION OF THE INVENTION

[0021]S. aureus is the most prevalent human pathogen in theStaphylococcal genus. It remains a major public health concern due toits tenacity, potential destructiveness and increasing resistance toantimicrobial agents. Much research has been focused on identifyinggenes or gene products of S. aureus which serve as targets in thedevelopment of new antibacterial agents.

[0022] Using transposon mutagenesis, a gene referred to herein as RAT(regulator of autolytic activity)(SEQ ID NO:1), which is specificallyinvolved in regulation of autolytic activity of S. aureus, has beenidentified. To identify the RAT gene, a Tn551 transposon library of S.aureus strain RN6390 was constructed. The library was screened for genesthat affected expression of genes encoding the capsular polysaccharide(cap genes—16 genes encoded within the cap operon) of S. aureus. Usingthe cap promoter linked to the GFP reporter gene (green fluorescentprotein), a mutant was identified that displayed significantly lower cappromoter activity. However, upon growing this mutant, it was discoveredthat this strain grew poorly in 03GL medium, reaching a maximum opticaldensity of 0.8 when the parental strain could achieve an OD650 nm of1.3. The defect was linked to the transposon insertion because thisphenotype could be back-crossed into the parental strain. The region ofthe mutant chromosome where the transposon was inserted was subsequentlysequenced; the sequence of the mutant locus is SEQ ID NO:3. This genewas designated RAT, or regulator of autolytic activity. The RAT mutantgene encodes a protein of 134 residues in length. The RAT mutant is atransposon mutant in which a Tn551 transposon is inserted at the 3′ endof the polynucleotide sequence, yielding a truncated protein orpolypeptide missing the last 13 amino acid residues of the wild-typeprotein. It is believed that the RAT mutant is a partial gene knockoutwhich enables the bacteria to survive. By “knock-out” it is meant thatan alteration in the target gene sequence occurs which results in adecrease of function of the target gene.

[0023] Zymogram analysis revealed that the RAT mutant strain displayedsignificantly enhanced autolytic activity as compared with the parent.This defect in autolytic activity was restored upon complementation ofthe mutant with a single copy of the RAT gene inserted into the mutantchromosome. RAT was cloned and sequenced (SEQ ID NO:1). RAT encodes a 17kDa protein of 147 residues in length. Forty-seven of the 147 residues(32%) are charged and the pI of RAT is predicted to be 7.38.

[0024] RAT plays a role in regulating autolytic activity of S. aureus.More specifically, in the presence of penicillin, the RAT mutant wasshown to readily increase lysis as compared to wild-type S. aureus.Furthermore, inactivation of the RAT locus was shown to render the S.aureus bacteria sensitive to lysis upon growth beyond the mid-log phase.To evaluate whether the cell lysis of the RAT mutant was additive to theeffect of a subinhibitory concentration of penicillin, 200 ng/ml ofpenicillin was added to a growing culture of the RAT mutant at an OD650nm of 0.5, corresponding to the mid-log phase. Contrary to the wild-typestrain that displayed no increase in optical density (i.e., no growth)upon addition of penicillin, the RAT mutant exhibited a furtherreduction in optical density as the growth cycle progressed (FIG. 1).This finding is consistent with the additive effect of penicillin uponthe lytic propensity of the RAT mutant late in the growth cycle. Similarresults were obtained using gentamicin and cephalothin. In plating thesecultures on agar plates without antibiotic selection, it was found thatthe RAT mutant has 1-2 log more kill than the parental strain. Incomparison to the parental strain without antibiotics, the RAT mutantwith subinhibitory concentrations of penicillin has a 3-4 log kill.

[0025] To analyze the autolytic activity of the RAT mutant, zymographicanalysis of cell-associated murein hydrolases was performed. Bacterialcells were centrifuged, washed, and resuspended in SDS-gel loadingbuffer, heated for 3 minutes at 100° C., recentrifuged, and thesupernatant applied to a SDS-gel containing heat-killed S. aureusRN4220. Following electrophoresis, the gel was soaked in 0.1 percentTriton X100 at 37° C. overnight to hydrolyze RN4220 cells that had beenattacked by autolytic enzymes in the cell extracts. After incubation,the gel was stained with one percent methylene blue and destained inwater. Clear bands, indicating zones of murein hydrolase activity werefound to be enhanced in the RAT mutant as compared with the wild-typecontrol. As a positive control, a sarA mutant was utilized. The sarAgene normally represses murein hydrolase activity.

[0026] To assess the viability of RAT mutant cells late in the growthcycle, the bacterial cells obtained from different parts of the growthcycle were stained with propidium iodide. Penetration of the cell withpropidium iodide indicates cell death or necrosis. Many of the RATmutant cells picked up the propidium iodide stain as the growth cyclelengthened (FIG. 2), thus accounting for the decrease in opticaldensities in the RAT mutant during the late log phase.

[0027] The cell wall morphology of the RAT mutant strain differs fromthe wild-type strain. RAT mutant cells, undergoing division, exhibited athicker cell wall than the wild-type strain. The outer contour of theRAT mutant was rough while the surface of the wild type cells wassmooth. Irregularities in the outer cell wall have previously beenassociated with altered autolytic activities in mutants. Bacteriumpossessing nucleotide sequences with a sufficient degree of homology toSEQ ID NO:3 also will exhibit an increase sensitivity to lysis.

[0028] The bacterial cell wall is maintained by competing enzymesinvolved in the synthesis and lysis of the cell wall. Examples ofautolytic enzymes include glucosamidase, muramidase, amidase, andendopeptidase. The synthesis of bacterial cell walls is a dynamicprocess requiring the precise regulation of both synthetic and autolyticactivities. The autolytic activity of many bacteria is carefullycontrolled during the growth cycle in particular by regulatory elements.A disruption of these regulatory elements alters autolytic activity andleads to premature cell lysis during growth. The RAT mutant has a defectin autolytic activity which prevents the mutant from reaching thestationary phase of growth. By northern blot analysis, it was shown thatthe RAT mutation had an effect on the expression of autolytic enyzmessuch as lytN, lytM, and atl. The novel cell wall hydrolase, lytN, andthe glycylglycine endopeptidase, lytM, are up-regulated by the RATmutation. Conversely, the RAT mutation down-regulated the regulators ofautolytic activity, namely, lytS, lytR, lrgA, lrgB, arlR, and arlS.Furthermore, the RAT mutation affects the expression of certain other S.aureus genes, e.g., hla, spa, abcA, scdA, pbp2, pbp4, and sspA. Both hlaand scdA expression are down-regulated by the RAT mutation, whereas bothspa and abcA expression are up-regulated by the RAT mutation.

[0029] In a comparison of the RAT gene sequence with the genomes ofother microbes, homologs with significant sequence similarity wereidentified. The RAT gene or a homolog thereof performs a role inregulating the autolytic activity of bacteria, including but not limitedto: Staphylococcus aureus (such as Staphylococcus aureus N315,Staphylococcus aureus strain Mu50, Staphylococcus aureus strain NCTC8325, Staphylococcus aureus MSSA strain, Staphylococcus aureus MRSAstrain, Staphylococcus aureus COL, Staphylococcus epidermidis, andStaphylococcus sciuri), Sinorhizobium species (e.g., meliloti), Listeriaspecies (e.g., monocytogenes), Clostridium species (e.g.,acetabutylicum, difficile), Vibrio species (e.g., cholerae),Corynebacterium species (e.g., diptheriae), Brucella species (e.g.,suis), Pseudomonas species (e.g., aeruginosa, syringae, putida),Shewanella species (e.g., putrefasciens), Mesorhizobium species (e.g.,loti), Caulobacter species (e.g., crescentus), Lactococcus species(e.g., lactis), Mycobacterium species (e.g., smegmatis, leprae,tuberculosis), Burkholderia species (e.g., mallei, pseudomallei),Geobacter species (e.g., sulfurreducens), Treponema species (e.g.,denticola), Bacillus species (e.g., stearothermophilus, anthracis,subtilis, halodurnas), Escherichia species (e.g., coli), Enterococcusspecies (e.g., faecalis), Salmonella species (e.g., dublin, enteriditis,paratyphi, typhi), Klebsiella species (e.g., pneumoniae), Bordetellaspecies (e.g., parapertussis), Actinobacillus species (e.g.,actinomycetemcomitans), Streptomyces species (e.g., coelicolor),Streptococcus species (e.g., pyogenes, pneumoniae) and Acinetobacterspecies.

[0030] Useful homologous sequences are those which encode a polypeptidewhich increases bacterial susceptibility to autolysis or increased lysisdue to antibiotic administration. In a preferred embodiment thepolynucleotide sequence is at least 40 percent homologous to the SEQ IDNO:1 or SEQ ID NO:3. In a more preferred embodiment the polynucleotidesequence is at least 60 percent homologous to the SEQ ID NO:1 or SEQ IDNO:3. In a most preferred embodiment the polynucleotide sequence is atleast 80 percent homologous to the SEQ ID NO:1 or SEQ ID NO:3.

[0031] The present invention includes the nucleic acid sequences for theRAT gene and RAT mutant gene and polypeptides encoded thereby. Forpurposes of the present invention, polypeptides encoded by the RAT geneand RAT mutant gene are referred to herein as RAT polypeptides and RATmutant polypeptides, respectively. Exemplary nucleic acid sequences ofthe present invention are SEQ ID NO:1 and SEQ ID NO:3. However, by theterm “nucleic acid sequence” it is meant to include any form of DNA orRNA such as cDNA or genomic DNA or mRNA, respectively, encoding a RATpolypeptide or RAT mutant polypeptide, or an active fragment thereofwhich are obtained by cloning or produced synthetically by well-knownchemical techniques. DNA may be double- or single-stranded.Single-stranded DNA may comprise the coding or sense strand or thenon-coding or antisense strand. Thus, the term nucleic acid sequencealso includes sequences which hybridize under stringent conditions tothe above-described polynucleotides. As used herein, the term “stringentconditions” means at least 60% homology at hybridization conditions of60° C. at 2×SSC buffer.

[0032] In a preferred embodiment, the nucleic acid sequence comprisesthe cDNA of SEQ ID NO:1or a homologous sequence or fragment thereofwhich encodes a polypeptide having similar activity to the polypeptide(SEQ ID NO:2) encoded by RAT. In another preferred embodiment, thenucleic acid sequence comprises the cDNA of SEQ ID NO:3 or a homologoussequence or fragment thereof which encodes a polypeptide having similaractivity to the polypeptide (SEQ ID NO:4) encoded by the RAT mutantgene. Due to the degeneracy of the genetic code, nucleic acid sequencesof the present invention may also comprise other nucleic acid sequencesencoding the RAT polypeptide or RAT mutant polypeptide and derivatives,variants or active fragments thereof. The present invention also relatesto variants of these nucleic acid sequences which may be naturallyoccurring, i.e., allelic variants, or mutants prepared by well knownmutagenesis techniques.

[0033] The present invention also relates to a conditional mutantwhereby the RAT gene or RAT mutant gene can be expressed under aninducible promoter.

[0034] The present invention also relates to vectors comprising nucleicacid sequences of the present invention and host cells which aregenetically engineered with these vectors to produce active RATpolypeptides or RAT mutant polypeptides, or fragments thereof.Generally, any vector suitable to maintain, propagate or express thenucleic acid sequences of this invention in a host cell may be used forexpression in this regard.

[0035] The nucleic acid sequences and polypeptides of the presentinvention, as well as vectors and host cells expressing the polypeptidesare useful as research tools to enhance the understanding of theautolytic process of S. aureus and other bacteria. The methods andcompositions of the present invention are believed to be effective inother bacteria having significant homology with the polynucleotidesequence of RAT or the RAT mutant.

[0036] Further, these compositions are useful in the identification ofagents which interact with either the RAT gene or RAT mutant gene, orpolypeptides encoded thereby to modulate autolytic activity of thebacteria. By “interact” it is meant that the agent increases ordecreases expression of the RAT gene or RAT mutant gene, or increases ordecreases activity of a RAT polypeptide or RAT mutant polypeptide. In apreferred embodiment, agents will decrease, interfere with or inhibitRAT gene or RAT mutant gene expression or decrease, interfere with orinhibit the activity of a RAT polypeptide or RAT mutant polypeptide sothat the bacteria is lysed more easily. Examples of such agents include,but are not limited to, antisense molecules or ribozymes targeted to theRAT gene or RAT mutant gene which inhibit the gene expression, means forintroduction of mutations into the RAT gene or RAT mutant gene whichinhibit gene expression or produce a polypeptide with decreasedactivity, and small organic molecules or peptides which are capable ofinhibiting activity of the RAT polypeptides or RAT mutant polypeptidesor the genes themselves (e.g., by binding to the promoter region of thegene to inhibit transcription and subsequent expression). The activesite of the RAT polypeptide, could be used to simulate activity in themutant. Alternatively, a small compound library can be used to screenfor agents which augment the lytic activity of the RAT gene or RATmutant gene. This augmentation of lytic activity can be monitored bybinding of the small compound to the RAT polypeptide or RAT mutantpolypeptide and determining the ability of the small compound to inducecell lysis.

[0037] Accordingly, one aspect of the present invention provides ananalog library to produce a very large number of potential molecules forregulating the RAT expression system, and in general the greater thenumber of analogs in the library, the greater the likelihood that atleast one member of the library will effectively regulate the RATexpression system. Designed libraries following a particular templatestructure and limiting amino acid variation at particular positions aremuch preferred, since a single library can encompass all the designedanalogs and the included sequences will be known and presented inroughly equal numbers. By contrast, random substitution at only sixpositions in an amino acid sequence provides over 60 million analogs,which is a library size that begins to present practical limitationseven when utilizing screening techniques as powerful as phage display.Libraries larger than this would pose problems in handling, e.g.,fermentation vessels would need to be of extraordinary size, and moreimportantly, the likelihood of having all of the planned polypeptidesequence variations represented in the prepared library would decreasesharply. It is therefore preferred to create a designed or biasedlibrary, in which the amino acid positions designated for variation areconsidered so as to maximize the effect of substitution on the RAT orRAT mutant regulation characteristics of the analog, and the amino acidresidues allowed or planned for use in substitutions are limited.

[0038] The use of replicable genetic packages, such as thebacteriophages, is one method of generating novel polypeptide entitiesthat regulate RAT or RAT mutant expression. This method generallyconsists of introducing a novel, exogenous DNA segments into the genomeof a bacteriophage (or other amplifiable genetic package) so that thepolypeptide encoded by the non-native DNA appears on the surface of thephage. When the inserted DNA contains sequence diversity, then eachrecipient phage displays one variant of the template (parental) aminoacid sequence encoded by the DNA, and the phage population (library)displays a vast number of different but related amino acid sequences.

[0039] Such techniques make it possible not only to screen a largenumber of potential binding molecules but make it practical to repeatthe binding/elution cycles and to build secondary, biased libraries forscreening analog-displaying packages that meet initial criteria.

[0040] It is well-known to those normally skilled in the art that it ispossible to replace peptides with peptidomimetics. Peptidomimetics aregenerally preferable as therapeutic agents to peptides owing to theirenhanced bioavailability and relative lack of attack from proteolyticenzymes. Accordingly, the present invention also providespeptidomimetics and other lead compounds which can be identified basedon data obtained from structural analysis of the RAT or the RAT mutant.A potential analog may be examined through the use of computer modelingusing a docking program such as GRAM, DOCK, or AUTODOCK. This procedurecan include computer fitting of potential analogs. Computer programs canalso be employed to estimate the attraction, repulsion, and sterichindrance of an analog to a potential binding site. Generally thetighter the fit (e.g., the lower the steric hindrance, and/or thegreater the attractive force) the more potent the potential drug will besince these properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a potential drug themore likely that the drug will not interfere with other properties ofthe RAT expression system. This will minimize potential side-effects dueto unwanted interactions with other proteins.

[0041] Initially a potential analog could be obtained by screening arandom peptide library produced by a recombinant bacteriophage, forexample, or a chemical library. An analog ligand selected in this mannercould be then be systematically modified by computer modeling programsuntil one or more promising potential ligands are identified.

[0042] Such computer modeling allows the selection of a finite number ofrational chemical modifications, as opposed to the countless number ofessentially random chemical modifications that could be made, and ofwhich any one might lead to a useful drug. Thus, the three-dimensionalstructure and computer modeling, a large number of compounds may berapidly screened and a few likely candidates may be determined withoutthe laborious synthesis of untold numbers of compounds.

[0043] Once a potential peptidomimetic or lead compound is identified itcan be either selected from a library of chemicals commerciallyavailable from most large chemical companies including Merck,GlaxoWelcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartisand Pharmacia UpJohn, or alternatively the potential ligand issynthesized de novo. As mentioned above, the de novo synthesis of one oreven a relatively small group of specific compounds is reasonable in theart of drug design.

[0044] Agents of the present invention may comprise antibodies againstthe RAT polypeptide or RAT mutant polypeptide. Antibodies against theRAT polypeptide or RAT mutant polypeptide can facilitate selectivedelivery of a cytotoxic agent to S. aureus or other bacteria.Alternatively, antibodies can serve as the agent, binding to the RATpolypeptide or RAT mutant polypeptide thereby inhibiting activity. TheRAT polypeptides or RAT mutant polypeptide, or epitope bearing fragmentsthereof can be used as immunogens to produce antibodies immunospecificfor such polypeptides. Various techniques well known in the art can beused routinely to produce antibodies (Kohler, G. and Milstein, C.,Nature 1975: 256: 495-497; Kozbor et al., Immunology Today, 1983: 4: 72;Cole et al., Monoclonal Antibodies and Cancer Therapy, 1985: pp 77-96).

[0045] Accordingly, the present invention also relates to agentsidentified as inhibitors of RAT gene or RAT mutant gene expression orRAT polypeptide or RAT mutant polypeptide activity and methods for usingthese agents to increase lysis of S. aureus and other bacteria, therebyinhibiting their growth and infectivity. These agents can beincorporated into a pharmaceutical composition and administered to ahost to inhibit growth and infectivity of S. aureus and other bacteriain the host. The term “host” as used herein includes humans.

[0046] Pharmaceutical compositions of the present invention comprise aneffective amount of an agent which inhibits the expression of the RATgene or RAT mutant gene or an activity of the RAT polypeptide or RATmutant polypeptide and a pharmaceutically acceptable vehicle. By“effective amount” it is meant an amount which inactivates the RAT geneor RAT mutant gene locus and renders S. aureus or other bacteriasusceptible to killing through cell lysis. The pharmaceuticalcompositions can be administered to a host, preferably a human, toinhibit the growth of S. aureus or other bacteria in the host. Thepharmaceutical composition can be administered alone, or in combinationwith an antibiotic such as penicillin, gentamicin and cephalothin toenhance killing or lysis of the bacteria. Pharmaceutical compositions ofthe present invention can be administered by various routes, including,but not limited to, topically, intramuscularly, intraperitoneally,intranasally, orally, subcutaneously, or intravenously.

[0047] The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the agents or pharmaceutical compositions of theinvention. The kit may be used for identifying the presence of RAT or aRAT mutant gene or RAT polypeptide or RAT mutant polypeptide in abiological sample by analyzing the sample for the presence of RAT or aRAT mutant. Detection of RAT or RAT mutant cells in a sample areindicative of the patient being susceptible to treatment for thebacterial infection using conventional antibiotic treatment, such aspenicillin. Associated with such container(s) can be a notice in theform of prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency or manufacture, use or sale for humanadministration. In addition, the polypeptides of the present inventionmay be employed in conjunction with other therapeutic compounds.

[0048] The embodiments herein described are not meant to be limiting tothe invention. Those of skill in the art will appreciate the inventionmay be practiced by using numerous chemical entities and by numerousmethods all within the breadth of the following claims.

1 4 1 444 DNA Staphylococcus aureus CDS (1)..(444) 1 atg tct gat caa cataat tta aaa gaa cag cta tgc ttt agt ttg tac 48 Met Ser Asp Gln His AsnLeu Lys Glu Gln Leu Cys Phe Ser Leu Tyr 1 5 10 15 aat gct caa aga caagtt aat cgc tac tac tct aac aaa gtt ttt aag 96 Asn Ala Gln Arg Gln ValAsn Arg Tyr Tyr Ser Asn Lys Val Phe Lys 20 25 30 aag tac aat cta aca taccca caa ttt ctt gtc tta aca att tta tgg 144 Lys Tyr Asn Leu Thr Tyr ProGln Phe Leu Val Leu Thr Ile Leu Trp 35 40 45 gat gaa tct cct gta aac gtcaag aaa gtc gta act gaa tta gca ctc 192 Asp Glu Ser Pro Val Asn Val LysLys Val Val Thr Glu Leu Ala Leu 50 55 60 gat act ggt aca gta tca cca ttatta aaa cga atg gaa caa gta gac 240 Asp Thr Gly Thr Val Ser Pro Leu LeuLys Arg Met Glu Gln Val Asp 65 70 75 80 tta att aag cgt gaa cgt tcc gaagtc gat caa cgt gaa gta ttt att 288 Leu Ile Lys Arg Glu Arg Ser Glu ValAsp Gln Arg Glu Val Phe Ile 85 90 95 cac ttg act gac aaa agt gaa act attaga cca gaa tta agt aat gca 336 His Leu Thr Asp Lys Ser Glu Thr Ile ArgPro Glu Leu Ser Asn Ala 100 105 110 tct gac aaa gtc gct tca gct tct tcttta tcg caa gat gaa gtt aaa 384 Ser Asp Lys Val Ala Ser Ala Ser Ser LeuSer Gln Asp Glu Val Lys 115 120 125 gaa ctt aat cgc tta tta ggt aaa gtcatt cat gca ttt gat gaa aca 432 Glu Leu Asn Arg Leu Leu Gly Lys Val IleHis Ala Phe Asp Glu Thr 130 135 140 aag gaa aaa taa 444 Lys Glu Lys 1452 147 PRT Staphylococcus aureus 2 Met Ser Asp Gln His Asn Leu Lys GluGln Leu Cys Phe Ser Leu Tyr 1 5 10 15 Asn Ala Gln Arg Gln Val Asn ArgTyr Tyr Ser Asn Lys Val Phe Lys 20 25 30 Lys Tyr Asn Leu Thr Tyr Pro GlnPhe Leu Val Leu Thr Ile Leu Trp 35 40 45 Asp Glu Ser Pro Val Asn Val LysLys Val Val Thr Glu Leu Ala Leu 50 55 60 Asp Thr Gly Thr Val Ser Pro LeuLeu Lys Arg Met Glu Gln Val Asp 65 70 75 80 Leu Ile Lys Arg Glu Arg SerGlu Val Asp Gln Arg Glu Val Phe Ile 85 90 95 His Leu Thr Asp Lys Ser GluThr Ile Arg Pro Glu Leu Ser Asn Ala 100 105 110 Ser Asp Lys Val Ala SerAla Ser Ser Leu Ser Gln Asp Glu Val Lys 115 120 125 Glu Leu Asn Arg LeuLeu Gly Lys Val Ile His Ala Phe Asp Glu Thr 130 135 140 Lys Glu Lys 1453 402 DNA Staphylococcus aureus CDS (1)..(402) 3 atg tct gat caa cat aattta aaa gaa cag cta tgc ttt agt ttg tac 48 Met Ser Asp Gln His Asn LeuLys Glu Gln Leu Cys Phe Ser Leu Tyr 1 5 10 15 aat gct caa aga caa gttaat cgc tac tac tct aac aaa gtt ttt aag 96 Asn Ala Gln Arg Gln Val AsnArg Tyr Tyr Ser Asn Lys Val Phe Lys 20 25 30 aag tac aat cta aca tac ccacaa ttt ctt gtc tta aca att tta tgg 144 Lys Tyr Asn Leu Thr Tyr Pro GlnPhe Leu Val Leu Thr Ile Leu Trp 35 40 45 gat gaa tct cct gta aac gtc aagaaa gtc gta act gaa tta gca ctc 192 Asp Glu Ser Pro Val Asn Val Lys LysVal Val Thr Glu Leu Ala Leu 50 55 60 gat act ggt aca gta tca cca tta ttaaaa cga atg gaa caa gta gac 240 Asp Thr Gly Thr Val Ser Pro Leu Leu LysArg Met Glu Gln Val Asp 65 70 75 80 tta att aag cgt gaa cgt tcc gaa gtcgat caa cgt gaa gta ttt att 288 Leu Ile Lys Arg Glu Arg Ser Glu Val AspGln Arg Glu Val Phe Ile 85 90 95 cac ttg act gac aaa agt gaa act att agacca gaa tta agt aat gca 336 His Leu Thr Asp Lys Ser Glu Thr Ile Arg ProGlu Leu Ser Asn Ala 100 105 110 tct gac aaa gtc gct tca gct tct tct ttatcg caa gat gaa gtt aaa 384 Ser Asp Lys Val Ala Ser Ala Ser Ser Leu SerGln Asp Glu Val Lys 115 120 125 gaa ctt aat cgc tta tta 402 Glu Leu AsnArg Leu Leu 130 4 134 PRT Staphylococcus aureus 4 Met Ser Asp Gln HisAsn Leu Lys Glu Gln Leu Cys Phe Ser Leu Tyr 1 5 10 15 Asn Ala Gln ArgGln Val Asn Arg Tyr Tyr Ser Asn Lys Val Phe Lys 20 25 30 Lys Tyr Asn LeuThr Tyr Pro Gln Phe Leu Val Leu Thr Ile Leu Trp 35 40 45 Asp Glu Ser ProVal Asn Val Lys Lys Val Val Thr Glu Leu Ala Leu 50 55 60 Asp Thr Gly ThrVal Ser Pro Leu Leu Lys Arg Met Glu Gln Val Asp 65 70 75 80 Leu Ile LysArg Glu Arg Ser Glu Val Asp Gln Arg Glu Val Phe Ile 85 90 95 His Leu ThrAsp Lys Ser Glu Thr Ile Arg Pro Glu Leu Ser Asn Ala 100 105 110 Ser AspLys Val Ala Ser Ala Ser Ser Leu Ser Gln Asp Glu Val Lys 115 120 125 GluLeu Asn Arg Leu Leu 130

What is claimed is:
 1. A nucleic acid sequence which regulatesexpression of polypeptides involved in autolytic processes in bacteria.2. The nucleic acid sequence of claim 1 wherein the bacteria isStaphylococcus aureus.
 3. The nucleic acid of claim 1 wherein thebacteria comprises Staphylococcus, Sinorhizobium, Listeria, Clostridium,Baciullus, Corynebacterium, Brucella, Pseudomonas, Shweanella,Mesorhizobium, Caulobacter, Lactococcus, Mycobacterium, Burkholderia,Geobacter, Treponema, Vibrio, Escherichia, Enterococcus, Salmonella,Klebsiella, Bordetella, Actinobacillus, Streptomyces, Streptococcus, orAcinetobacter.
 4. A nucleic acid sequence comprising SEQ ID NO:1 or SEQID NO:3.
 5. A polypeptide encoded by the nucleic acid sequence ofclaim
 1. 6. A polypeptide encoded by the nucleic acid sequence of SEQ.ID NO: 1 or SEQ ID NO:3.
 7. A polypeptide sequence comprising SEQ ID NO:2 or SEQ ID NO:4.
 8. A composition comprising a selected transposon andthe nucleic acid sequence of claim
 1. 9. A vector comprising the nucleicacid sequence of claim
 1. 10. A host cell comprising the vector of claim9.
 11. The polypeptide of claim 5 wherein the bacteria comprisesStaphylococcus aureus.
 12. The polypeptide of claim 5 wherein thebacteria comprises Staphylococcus, Sinorhizobium, Listeria, Clostridium,Baciullus, Corynebacterium, Brucella, Pseudomonas, Shweanella,Mesorhizobium, Caulobacter, Lactococcus, Mycobacterium, Burkholderia,Geobacter, Treponema, Vibrio, Escherichia, Enterococcus, Salmonella,Klebsiella, Bordetella, Actinobacillus, Streptomyces, Streptococcus, orAcinetobacter.
 13. A method for identifying agents which inhibit growthand infectivity of bacteria comprising identifying agents which inhibitexpression of a nucleic acid sequence of claim 1 or activity of apolypeptide encoded thereby.
 14. The method of claim 13 wherein thebacteria is Staphylococcus aureus.
 15. The method of claim 13 whereinthe bacteria comprises Staphylococcus, Sinorhizobium, Listeria,Clostridium, Baciullus, Corynebacterium, Brucella, Pseudomonas,Shweanella, Mesorhizobium, Caulobacter, Lactococcus, Mycobacterium,Burkholderia, Geobacter, Treponema, Vibrio, Escherichia, Enterococcus,Salmonella, Klebsiella, Bordetella, Actinobacillus, Streptomyces,Streptococcus, or Acinetobacter.
 16. A method of inhibiting growth andinfectivity of bacteria comprising contacting the bacteria with an agentwhich inhibits the expression of a nucleic acid sequence of claim 1 orthe activity of a polypeptide encoded thereby.
 17. The method of claim16 wherein the bacteria is Staphylococcus aureus.
 18. The method ofclaim 16 where the bacteria comprises Staphylococcus, Sinorhizobium,Listeria, Clostridium, Baciullus, Corynebacterium, Brucella,Pseudomonas, Shweanella, Mesorhizobium, Caulobacter, Lactococcus,Mycobacterium, Burkholderia, Geobacter, Treponema, Vibrio, Escherichia,Enterococcus, Salmonella, Klebsiella, Bordetella, Actinobacillus,Streptomyces, Streptococcus, or Acinetobacter.
 19. A pharmaceuticalcomposition for use as an antibacterial agent comprising apharmaceutically acceptable vehicle and either an agent which inhibitsthe expression of a nucleic acid sequence of claim 1 or an agent whichinhibits the activity of a polypeptide encoded thereby.
 20. Thepharmaceutical composition of claim 19 wherein the bacteria isStaphylococcus aureus.
 21. The pharmaceutical composition of claim 19wherein the bacteria comprises Staphylococcus, Sinorhizobium, Listeria,Clostridium, Baciullus, Corynebacterium, Brucella, Pseudomonas,Shweanella, Mesorhizobium, Caulobacter, Lactococcus, Mycobacterium,Burkholderia, Geobacter, Treponema, Vibrio, Escherichia, Enterococcus,Salmonella, Klebsiella, Bordetella, Actinobacillus, Streptomyces,Streptococcus, or Acinetobacter.
 22. A kit for identifying the presenceof RAT polypeptides or comprising a means for analyzing a biologicalsample for the presence of a RAT polypeptide, whereby detection of a RATpolypeptide in the sample is indicative of the susceptibility totreatment for a bacterial infection.
 23. A kit for identifying thepresence of the RAT gene comprising a means for analyzing a biologicalsample for the presence of the RAT gene.
 24. A kit for identifying thepresence of RAT mutant polypeptides or comprising a means for analyzinga biological sample for the presence of a RAT mutant polypeptide,wherein detection of a RAT mutant polypeptide in the sample isindicative of the susceptibility to treatment for a bacterial infection.25. A kit for identifying the presence of the RAT mutant gene comprisinga means for analyzing a biological sample for the presence of the RATmutant gene.